To investigate the anti-inflammatory effects of high-density lipoprotein (HDL) in mice with rheumatoid arthritis (RA) induced by collagen.Male DBA/1 mice (8-week-old) were divided into three groups: control , collagen-induced arthritis (CIA), and CIA + HDL. CIA was induced with bovine type II collagen, and after the injection of bovine type II collagen, the CIA + HDL group received an injection of HDL on day 28 followed by HDL injections four times every 3 days. Mice were weighed, the paws were scored, and paw thickness was measured beginning on day 21. Additionally, the levels of tumor necrosis factor-alpha (TNF-α) and IL-6 were measured by ELISA kits, tissue sections of paws were stained with hematoxylin and eosin, and the inflammatory signaling pathway was analyzed by western blotting.We found that the production of pro-inflammatory cytokines TNF-α and IL-6 in mice which received HDL decreased 45.14 and 35.02%, respectively. And we also found that HDL could significantly decrease the level of anti-type-II-collagen IgG2a and inhibit the neutrophil infiltration and cell proliferation and protect the ankle joint from type II collage-induced injury. Western blot analysis indicated that HDL could also inhibit the activation of the NF-κB, MAPK, and ERK signaling pathways in RA mice.HDL can inhibit the inflammation induced by bovine type II collagen and the development of RA. HDL can downregulate the expression of toll-like receptor-induced pro-inflammatory cytokines, such as TNF-α, IL-6, and endothelial cell vascular cell adhesion molecule 1, and can increase endothelial nitric oxide synthase production. HDL has also been shown to reduce monocyte CD11b expression and migration along a monocyte chemotactic protein-1 gradient. These potent anti-inflammatory properties of HDL may be critical for protection against other inflammatory diseases , as well as the mortality rates caused by endotoxin (lipopolysaccharide), were reduced in transgenic mice in which plasma levels of high-density lipoproteins (HDLs) were twofold higher than normal  is a systemic inflammatory autoimmune disease that causes joint destruction, as evidenced by radiological findings, such as joint space narrowing and bone erosion, which are related to functional disability. Both B cells and T cells aggregate in the synovium of inflamed joints and mediate the pathogenesis of RA. Studies have shown that an increase of proinflammatory cytokines, such as IL-6 and TNF-α, is critically associated with the histological characteristics of arthritis –6. Humanin vitro . Based oImmunization-grade bovine type II collagen solution was purchased from Chondrex, Inc. Freund’s complete adjuvant (FCA) was purchased from Sigma. ELISA kits were purchased from Yuan Ye Technologies. Human HDL came from ProSpec-Tany Technogene Ltd. The p44/42 MAPK (Erk1/2), P-p44/42 MAPK (Erk1/2), P-IκBα, p38, P-p38, and NF-κB p65 primary antibodies were purchased from CST. The GAPDH secondary antibody was purchased from Epitomics, and the lamin B secondary antibody was from Santa Cruz.Eight-week-old male DBA/1 mice were purchased from the Beijing Vital River Laboratory Animal Technology Co. and maintained under specific bacteria-free conditions. All experiments were approved by the Animal Use Committee of the affiliated hospital of Qingdao University. The mice were divided into three groups: control , CIA, and CIA + HDL. The CIA and CIA + HDL groups were induced according to a previous report . On day Blood was collected from the retro-orbital sinus of the mice at the end of the study. TNF-α, IL-6 levels were measured using ELISA kits according to the manufacturer’s instructions.The serum level of anti-collagen-II antibody IgG2a was determined by an ELISA method as described . BrieflyMice were sacrificed at day 60, and the paws were fixed in 10% neutral buffered formalin, decalcified by immersion in 10% EDTA solution for 1 month, and embedded in paraffin. Tissue sections were stained with H&E. Images were captured with a Nikon TE2000-S  and analyzed.Mice were sacrificed at day 60, the ankle joint was separated, proteins were extracted from the tissue, and protein concentration was measured using a BCA assay kit. Equal amounts of protein per sample were loaded on 12% pre-cast SDS-PAGE gels with MOPs buffer, and following protein separation, proteins were transferred to PVDF membranes. Membranes were incubated for 2 h at room temperature with 5% BSA in Tris buffer and overnight at 4°C with specific primary antibodies. Next, membranes were washed with TBST, incubated with secondary antibodies for 1 h, and rewashed more than three times. Finally, immune reactivities were visualized and scanned into a computer. Individual bands were analyzed with Image J software.p < 0.05 was considered to be statistically significant.Data were analyzed using one-way analysis of variance followed by least significant difference determination using SPSS 22.0 software for Windows . A value of Changes in the body weights of the mice in each group showed that the CIA model group and the CIA + HDL treatment group began to decrease on day 28. In Figure Inflammatory factors TNF-α and IL-6 reflect the arthritic development stage of CIA mice. As depicted in Figure p < 0.05) (Figure To determine the effect of HDL on anti-collagen-II antibody, the level of anti-collagen-II IgG2a in serum was measured by ELISA. And the result showed that the level of IgG2a in CIA + HDL group were significantly lower than that of CIA  Figure .We performed HE staining to further assess ankle joint inflammation induced by collagen. Compared with the control group, the joints of the mice in the CIA group exhibited significant pathological changes, including neutrophil infiltration, cell proliferation, and disorderly cell arrangements. After treatment with HDL, we observed that although there was limited cell proliferation and inflammatory cell infiltration, the pathological changes were significantly reduced in comparison with untreated mice Figure A. As FigTo investigate the potential mechanisms of the effects of HDL on RA induced by collagen, we collected the sample from joint tissue, and then examined the expression of proteins in inflammatory signaling pathways. As shown in Figure in vivo data have confirmed earlier in vitro findings that show regulation of many cytokines such as IL-6, IL-8 expression in RA synovial tissue by neutralizing TNF-α activity (Cytokines and other inflammatory molecules can be detected in the joints of RA patients. IL-1, IL-6, and other cytokines have been proposed to explain the mechanism of inflammation , 13. Speactivity . Thus, tactivity . Based oactivity . We alsoactivity , 18. Incactivity  in RA miAdditionally, we explored the effect of HDL on the inflammatory signaling pathway in CIA mice. Western blotting results showed that expression levels of ERK1/2 and p38MAPK in the MAPK pathway and p-IκB and p65 in the NF-κB pathway were reduced after treatment with HDL. Thus, HDL can inhibit the activity of inflammatory signaling pathways in RA mice, which is consistent with previous studies –21.In addition, besides the reduced TNF-α expression and activation of inflammatory signaling pathway, S1P may also play an important role in therapeutic effects of HDL in RA. ApoM-bound S1P is a key component of HDL and is responsible for several HDL-associated protective functions . Many stin vivo, including decreased expression of inflammatory factors and reduced activity of inflammatory signaling pathways. Thus, HDL has the potential therapeutic effects in RA.In conclusion, we analyzed the effect of HDL on CIA in mice. We observed that HDL was associated with reduced inflammatory RA conditions All experiments were approved by the Animal Use Committee of the affiliated hospital of Qingdao University.YW and BL conceived and designed the experiments. YW, SL, GZ, SW, YY, and QD performed the experiments. SL and YW analyzed the data and wrote the paper. YW and BL contributed reagents/materials/analysis tools.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Here we have identified and explored the role of Sof-dac, Sof-six1/2, Sof-eya in eye morphogenesis, and nervous structures controlling the visual function in Sepia officinalis. We compare that with the already shown expressions in eye development of Sof-otx and Sof-pax genes. Rhodopsin is the pigment responsible for light sensitivity in metazoan, which correlate to correlate visual function and eye development. We studied Sof-rhodopsin expression during retina differentiation. By in situ hybridization, we show that (1) all of the RDGN genes, including Sof-pax6, are expressed in the eye area during the early developmental stages but they are not expressed in the retina, unlike Sof-otx, which could have a role in retina differentiation; (2) Sof-rhodopsin is expressed in the retina just before vision gets functional, from stage 23 to hatching. Our results evidence a role of Sof-six1/2, Sof-eya, and Sof-dac in eye development. However, the gene network involved in the retinal photoreceptor differentiation remains to be determined. Moreover, for the first time, Sof-rhodopsin expression is shown in the embryonic retina of cuttlefish suggesting the evolutionary conservation of the role of rhodopsin in visual phototransduction within metazoans. These findings are correlated with the physiological and behavioral observations suggesting that S. officinalis is able to react to light stimuli from stage 25 of organogenesis on, as soon as the first retinal pigments appear.In metazoans, there is a remarkable diversity of photosensitive structures; their shapes, physiology, optical properties, and development are different. To approach the evolution of photosensitive structures and visual function, cephalopods are particularly interesting organisms due to their most highly centralized nervous system and their camerular eyes which constitute a convergence with those of vertebrates. The eye morphogenesis in numerous metazoans is controlled mainly by a conserved Retinal Determination Gene Network (RDGN) including  It includes pax6, eya (eyes absent), six (sine oculis), and dac (dachshund) which act as a regulatory network of eye formation and retinal differentiation  and Notch play a key role in photoreceptor cell differentiation and retinal organization  induces a transduction cascade that results in the opening or closing of cation cGMP-gated channels in the photoreceptors  . Expression of other genes of the RDGN, six, eya, and dac has been described in the central nervous system, optic area and light organ of E. scolopes and during eye morphogenesis of the D. pealeii embryo . The protocols for the staging and the fixation of the embryos are detailed in Buresi et al.  and the French law (decree 87/848) regulating animal experimentation that does not concerns embryos before hatching. Nonetheless, all experiments were performed according to France and European ethical guidelines in the treatment and handling of all animals used within this study. Fertilized eggs of i et al. .Sof-six1/2, Sof-eya, Sof-dac, and Sof-opsin were characterized in an embryonic EST library of S. officinalis  and Sof-rhodopsinR3 (5′-CGCCGATGAAGCCGTATACT-3′), Sof-six1/2F3 (5′-CCTCCCATGCTTCCATCGTT-3′) and Sof-six1/2R3 (5′-GAAATTTTCGGCGGACCCTG-3′), Sof-eyaF1 (5′-ACCTACACGAGGTGGTCGTC-3′), and Sof-eyaR1 (5′-CCACGGACTCCAGTTGCTAT-3′), Sof-dacF1: (5′-CGGCCAGAAGCACCAGTTAT-3′) and Sof-dacR1 (5′-CAGTGCTTCACCATTGGGGACT-3′). They were used to amplify respectively a 311, 346, 372, and 400 bp fragment. Sof-dac was cloned as described in the protocol of Buresi et al. . The probes for in situ hybridization were synthetized from theses plasmids.mRNA fragments of a et al. . From thi et al. . ConcernSof-six1/2, Sof-eya, Sof-dac, and Sof-opsin gene transcripts during early embryogenesis of S. officinalis (from stage 18 to stage 22) were examined by whole-mount in situ hybridization (ISH) according to the protocol detailed in Buresi et al. (RNA probes were synthetized with the digoxigenin (DIG) RNA labeling mix from Roche . According to the sense of PCR product insertion into the vector, sense and antisense probe were obtained with T3 and T7 polymerase (Roche). Spatio-temporal expression patterns of i et al. .in situ hybridization were impregnated in 0.12M phosphate buffered  plus 30% sucrose treated with 0.1% DEPC (Diethyl pyrocarbonate) for 48 h at 4°C. Then, they were included in Tissue-Tek and blocks were frozen in isopentane cooled at −80°C for 1 min. Sections of 20 μm were performed with cryostat Leica and used for ISH experiments. From stage 23 to stage 30, the expression patterns of those genes were stained by a cryo-sections in situ hybridization. Note that control negatives were used for each slide as a test for the same embryo selected and each gene studied. Unless otherwise specified, all steps of the experiments were performed in a humid chamber at room temperature. After 30 min at room temperature, the sections were rehydrated 2 times in 1X phosphate buffered saline (1X PBS) with 0.1% DEPC and treated 1 time in standard 5X saline citrate  each time for 15 min. A prehybridization step was done in hybridization solution (HS) for 2 h at 65°C . Sections were next incubated overnight at 65°C in HS containing 300 ng/ml of probes. Excess probe was removed by 2 rinses in standard 2X saline citrate  . Slides were washed for 1 h at 65°C in standard 0.1X saline citrate . Sections were then treated twice (15 min each) with MABT . Saturation was performed for 1 h in blocking solution , 15% fetal bovine serum), followed by incubation for 1 h at 4°C with anti-digoxigenin antibodies (Roche) coupled to alkaline phosphatase (AP) and diluted at 1:500 in blocking solution . Excess antibody was eliminated by 3 rinses (10 min each) in MABT then 3 rinses (5 min each) in PTW (PBS plus 0.10% Tween20). Sections were impregnated for 20 min in AP solution  with 100 μM levamisole hydrochloride . The revelation of AP activity was conducted in AP solution  containing 165 μg/ml BCIP  and 330 μg/ml NBT (nitro-blue tetrazolium chloride) (Roche). The reaction was stopped by washing 3 times (10 min each) in 1X PBS. The slides were treated with DAPI .From stage 23 on, it is necessary to observe the expression on sections to localize the expressing cells. Embryos used for cryo-sections in situ hybridization, a POD-coupled anti-digoxigenin antibody (Roche) diluted 1:500 was used and bound antibodies were revealed using FITC-tyramide diluted 1:200 in PTW containing 0.001% of hydrogen peroxide, at room temperature for thrice 45 min, in the dark. After washing, the sections were mounted in Mowiol.For fluorescent in situ hybridizations. For cryo-sections ISH, the sections were observed under a Leica DMLB compound microscope. All images were taken by a camera color CoolSnapPro and treated using Adobe Photoshop Elements 9  for contrast and brightness.A Leica M16 2F binocular stereomicroscope was used to observe the embryos labeled by S. officinalis has long been the subject of morphological research , differentiation of two cell types begins. At stage 24, the retina begins to be slightly colored with orange in the periphery of the middle retina. From stage 25 on, the eyes are entirely orange and darken until hatching. Further in the development (stages 25 and 26), a third ectodermal fold covering the eye appears to form a secondary cornea . For both genes (Sof-six1/2 and Sof-eya), no expression is observed in the retina during these stages and from stage 23 to hatching, no expression is detected in any other tissues. By contrast, Sof-otx is expressed in the eye area as shown in whole-mount embryo ISH and this expression is located precisely in the retina at stage 20 and 21  and subesophageal (SBM) masses ,  Figures . By ISH  Figures . No expr to 30,   Figures . At stag Figures . From st Figures .Sof-rhodopsin expression is detected only in the undifferentiated retina  in S. officinalis. Surprisingly, no Sof-six1/2 and Sof-eya expression is shown in the eye area before stage 20 and after stage 22. Eya and Six have been strongly proposed to have an ancient role in eye development and their orthologs are involved in visual system development in both “invertebrates” and vertebrates  is expressed in S. officinalis embryo all along the development in the retina cells from the first steps of specification until the rhabdomeric photoreceptors are fully differentiated. Nevertheless, other genes appears to have a role in retina differentiation, such as Sof-otx, which is expressed in the retina as already shown  with Rx activate the expression of c-opsin, characteristic of ciliary photoreceptors  when the photoreceptor's differentiation started but its expression is not found from stage 26 when the retinal cell type organization is being achieved  is congruent with the upstream regulation of rhodopsin in vertebrates . We cannot exclude that they are expressed before stage 17 but as there are undifferentiated cells until stage 21, their role in the retina cell differentiation would be questioned. These findings reveal or reinforce the divergence and the broad complexity in the genetic network underlying the cephalopod retinal differentiation. Sof-six1/2, Sof-eya, and Sof-dac are localized only in the eye area questioning about the gene network involved in the differentiation of rhabdomeric photoreceptors in cephalopods. Actually, all of the previous studies in eye development in cephalopods have been performed by whole mount in situ hybridization. However, according to our results, we point out that gene expressions must be studied on sections to make it possible to exactly locate the expression of these genes at the cellular level. Nevertheless, cephalopods are an interesting model to study the evolution of the nervous system, the eye development complexity and the diversity of photosensitive structures. Besides the fact that RDGN expression levels could be outside the threshold of detection, the Sepia's retina development and the final differentiation of rhabdomeric photoreceptors is probably controlled by other genes than those identified until now. Thus, it will be necessary to explore the role of other genes such as Notch that is known to intervene in the retina and lens formation in vertebrates and Drosophila where it regulates the cell cycle progression within retina and lens  which has been consulted. LB-P is a member of Cuvier Commitee.Conceived of project: LB-P; Designed experiments: LB-P, BI, and AA; Executed experiments: BI and AA; Analyzed data: BI; Phylogenetic analysis: PL; Edited manuscript: BI, LB-P, PL, AA and YB.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


There was no difference between ASIC1a-deficient mice and wildtype controls. We are therefore not able to confirm that ASIC1a are involved in MPTP toxicity. The difference might relate to the subacute MPTP model we used, which more closely resembles the pathogenesis of Parkinson disease, or to further targets of amiloride.Inflammation contributes to the death of dopaminergic neurons in Parkinson disease and can be accompanied by acidification of extracellular pH, which may activate acid-sensing ion channels (ASIC). Accordingly, amiloride, a non-selective inhibitor of ASIC, was protective in an acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson disease. To complement these findings we determined MPTP toxicity in mice deficient for ASIC1a, the most common ASIC isoform in neurons. MPTP was applied i.p. in doses of 30 mg per kg on five consecutive days. We determined the number of dopaminergic neurons in the  The cause of cell death in PD is not known and there is currently no causal treatment for PD. Consequently, diverse changes observed in PD are investigated for their potential as a base for neuroprotective treatment strategies.Parkinson disease (PD) is a neurodegenerative movement disorder characterized by progressive degeneration of dopaminergic neurons in the vice versa [Inflammation is an important part of the PD pathogenesis. Activation of microglia and increased pro-inflammatory cytokines have been described in PD patients –5 and ince versa . Accordice versa . This inAdministration of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induces a PD-like pathology in several species, including mice and primates , 11. TheAcid-sensing ion channels (ASICs) are neuronal, proton-gated cation channels activated by low extracellular pH , 14. ASIGiven the evidence for tissue acidification in PD, it is plausible to hypothesize that ASICs might be involved in the pathogenesis of PD. Two studies indeed found that pharmacological inhibition of ASICs reduced neurodegeneration in animal models of PD. In an acute MPTP mouse model, degeneration of dopaminergic neurons was reduced by the nonselective ASIC inhibitor amiloride. Amiloride reduced the degeneration of dopaminergic axon terminals in the striatum and the degeneration of dopaminergic neurons in the SNc in a dose-dependent fashion . The morTo complement these findings with pharmacological inhibitors, we determined MPTP toxicity in mice deficient for ASIC1a, the most important ASIC subunit in neurons of the brain.ad libitum. The animal experiments were approved by the Animal Care Committee of the RWTH Aachen University and by the District Government of North Rhine Westphalia in Recklinghausen, Germany (No 84–08.05.2013.A056). For all experiments male 10-12-week old ASIC-1a null mutants (ASIC1a-/-) and wildtype littermates (ASIC1a+/+) were used. The generation of the ASIC1a-/- mice was described previously [-/- mice were backcrossed to C57BL/6 background for more than 8 generations. Mice received either MPTP hydrochloride in 0.9% saline or saline alone. MPTP was administered at a dose of 30 mg free base per kg body weight i.p. at 24 h intervals over 5 consecutive days. MPTP handling and safety measures were in accordance with published guidelines [All mice were housed and handled according to guidelines from the Federation for European Laboratory Animal Science Associations (FELASA). Mice were housed in a pathogen-free facility in a temperature-controlled room 20–24°C) with a 12 h light/dark cycle and with food and water –24°C witidelines . During 2O2 in TBS-T for 30 minutes followed by three washing steps with TBS-T. The primary anti-TH antibody  was incubated overnight at 4°C in a dilution of 1:1,000 in TBS-T containing 3% normal goat serum (Vector Laboratories). The sections were washed again with TBS-T and secondary antibody  was incubated in a dilution of 1:200 in TBS-T for 30 minutes. Subsequently, the sections were washed and incubated with Avidin-Biotin Complex (Thermo Fisher) for 30 minutes, followed by an additional washing step. Visualization of antibody binding was performed via diaminobenzidine  in a dilution of 1:20 in PBS for 10 minutes. Sections were mounted on microscope slides. Nissl-counterstaining was performed with haemalaum (VWR) and after dehydration sections were coverslipped with Entellan (Merck Millipore).The posterior parts of the brains, including the SN, were serially cut into 30 μm coronal sections. Every third section spanning the SN was stained for tyrosine hydroxylase (TH). The free-floating brain sections were washed three times in Tris-buffered saline (TBS) with 0.1% TritonX (TBS-T). Endogenous peroxidase was blocked to reduce unspecific background by incubation with 0.3% HTH-positive (dopaminergic) and Nissl-positive  cells in the lateral SNc of the right hemisphere were stereologically counted using the optical fractionator method  as described previously . In brieCatecholamines were measured by HPLC with electrochemical detection as described previously –29. StriData are presented as mean ± SEM with "n" equal to the number of animals. Statistical analyses were performed using GraphPad Prism 5.0 (GraphPad Software). The statistical test for comparison of treatment and genotype was 2-way ANOVA followed by Bonferroni posthoc test or by unpaired t-test as indicated in the text. The null hypothesis was rejected at the 0.05 level.-/- and ASIC1a+/+ mice not receiving MPTP. There was no difference in the number of dopaminergic neurons in the SNc, as identified by staining for tyrosine hydroxylase (TH), between the two genotypes  [Amiloride not only blocks ASICs, but also a number of other membrane proteins that might be involved in neurodegeneration in the MPTP mouse model, including the Naer (NCX) . NCX couer (NCX) –33 and ter (NCX) . Indeed,er (NCX) . Yet, ther (NCX) .Furthermore, the discrepancy may result from differences between the PD models used. Arias and colleagues used a single dose of MPTP (40 mg/kg) administered two hours after amiloride, whereas a subacute MPTP regimen (5x30 mg/kg) was used in this study. Oxidative stress, excitotoxicity and non-apoptotic cell death are more pronounced in the single dose MPTP paradigm , 37, whe


Spiritual well-being has become an increasingly important issue for the elderly people. The 12-item Spirituality Index of Well-Being (SIWB) is a well-validated instrument for assessing a patient’s current spiritual state. However, the psychometric properties of the SIWB in the Chinese elderly populations are not known. Therefore, this study translated the SIWB into Chinese and evaluated its psychometric properties.The English version of the SIWB was first translated into Chinese based on the Brislin’s translation model. The psychometric properties of the translated version of the SIWB (SIWB-C) was evaluated in 416 elderly Taiwanese recruited using a purposive sampling procedure from a medical center, a long-term care institution, and a community health center. Convergent validity was accessed using Pearson’s correlation coefficients of the SIWB-C, the EQ-5D-3 L health-related quality of life scale, and the Geriatric Depression Scale-5 (GDS-5). Exploratory factor analysis with Varimax rotation was performed to determine the construct validity. Confirmatory factor analysis was conducted for verification of the quality of the factor structures and demonstrating the convergent validity of the SIWB-C. An internal consistency test based on the Cronbach’s alpha coefficient and a stability test based on the Guttman split-half coefficient were also performed. Test-retest reliability was evaluated with intraclass correlation coefficient.p < 0.01) and 0.45 (p < 0.05), respectively. The intraclass correlation coefficient of the SIWB-C over a test-retest interval of two weeks was 0.989.Exploratory factor analysis confirmed the original two-dimensional structure of the scale. Confirmatory factor analysis indicated a well-fitting model and a fine convergent validity of the SIWB-C. The Cronbach’s alpha coefficient and the Guttman split-half coefficient for the SIWB-C were 0.94 and 0.84, respectively. The correlations between the SIWB-C with EQ-5D-3 L and GDS-5 were 0.22  contains supplementary material, which is available to authorized users. In Taiwan, the senior citizens accounted for 11.8% of the total population [Gomez and Fisher  proposedr = −0.35), EuroQol (r = 0.18), the Physical Functioning Index from the Short Form 36 (r = 0.28), and the Years of Healthy Life Scale (r = −0.35) [Given the importance of assessing spiritual well-being, several instruments have been developed for its measurement. In a systematic review, Monod et al. reviewed five instruments designed to assess spiritual well-being in clinical health research setting . The aut= −0.35) . In addi= −0.35) . The SIW= −0.35) . HoweverUsing a purposive sampling procedure, potential participants were recruited from three different settings: a medical center with 60 geriatric beds, a long-term care institution with 256 elderly residents, and a community health center with approximately 50 elderly visiting each day. They were identified by their treating physicians, health providers, and the community health center. The inclusion criteria included age of 65 years and older, the ability to communicate in Chinese, and had no obvious cognitive impairment as determined by the two research nurses of this study. The study was approved by the Institutional Review Board of the Taichung Veterans General Hospital, Taiwan (No. CE14109). Informed written consent was obtained from all participants.http://www.annfammed.org/content/suppl/2004/10/04/2.5.499.DC1/Daaleman_Appendix.pdf.The SIWB was developed to measure one’s perceptions of their spiritual quality of life. Based on the results of a factor analysis on 277 community-dwelling elderly individuals recruited from primary care clinic sites in the Kansas City metropolitan area, the initial 40-item version of the SIWB was reduced to 12 items, with a Cronbach’s alpha of 0.87 . The SIWThe Geriatric Depression Scale-5 (GDS-5) is a useful screening tool with five items for detecting geriatric depression . The fivThe EQ-5D-3 L is a measure of self-reported health status developed by the EuroQol Group. It consists of two components: a descriptive classification component and a Visual Analogue Scale (VAS). The descriptive component consists of five dimensions of health, including mobility, self-care, usual activities, pain/discomfort, and anxiety/depression. Each dimension has three levels, namely “no problems”, “some problems”, and “extreme problems” , 17. PerLinguistic validation of the Chinese language SIWB was performed using a standard procedure of translation and blind back-translation. Based on the Brislin’s translation model , Phase IPhase II was the psychometric testing of the SIWB-C obtained from phase I on Chinese elderly. Participants were recruited from three different settings: a medical center, a long-term care institute, and a community health center in Taiwan.Two trained research nurses were responsible for administering the questionnaires and collection data from the participants in the three locations: a hospital, a long-term care institution, and a community center. Participants were asked to complete a battery of self-report questionnaires, which consisted of the SIWB-C, the EQ-5D-3 L, the GDS-5 scales, and a questionnaire on socio-demographic information. For participants with low literacy level, each question was read aloud with explanation, if necessary, by the trained research nurses. The verbal responses of the participants were then recorded on the questionnaires. Test-retest reliability was evaluated by administering the SIWB-C to all the participants for a second time one week after the initial administration.Data were analyzed using IBM SPSS Statistics software package, version 20.0 for Windows . Categorical data were expressed as frequency and percentages. Continuous data were expressed as mean and standard deviation. In addition, face validity of the SIWB-C was tested on nine elderly with an educational level of elementary school. Convergent validity was accessed using Pearson’s correlation coefficients of the SIWB-C, the EQ-5D-3 L, and the GDS-5. The latter two measures were selected for assessing the convergent validity in this study following the original study of the SIWB.Exploratory factor analysis with Varimax rotation was performed to determine the construct validity. The reliability of the SIWB-C was tested with an internal consistency test based on the Cronbach’s alpha coefficient and a stability test based on the Guttman split-half coefficient. Test-retest reliability was analyzed with intraclass correlation coefficient using a two-way mixed effects model and type consistency.Confirmatory factor analysis was conducted using AMOS, version 22.0  with a maximum likelihood estimate. Average variance extracted (AVE)  were included for evaluating the convergent validity of the SIWB-C. Structural equation modeling was used to assess the fit 2-factor model compared with the 1-factor model. The goodness-of-fit of the two models were compared using absolute fit index, relative fit index, and parsimony fit index.A total of 416 elderly participants from three different settings were included in this study . Of the 416 participants, 287 (69%) were male and 129 (31%) were female. Their mean age was 81.1 ± 8.4 years. The basic characteristics of the participants are summarized in Table Face validity, construct validity, and convergent validity were used for the validation of the SIWB-C. To assess face validity, the SIWB-C was given to nine elderly with the educational level of elementary school to understand how they perceived and interpreted the items. The participants reported that the wordings of the SIWB-C were clear and they had little difficulty understanding it.p < 0.01) and 0.45 (p < 0.05), respectively (Table The construct validity of the SIWB-C was evaluated using exploratory factor analysis. The analysis confirmed the two-dimensional structure of the scale, with factor loadings above 0.30 on all items. The model could explain 68.6% of the total variance. Regarding the convergent validity of SIWB-C, the correlations of SIWB-C with EQ-5D-3 L and GDS-5 were 0.22 (The Cronbach’s alpha coefficient for the 12 items of the SIWB-C was 0.94. For the self-efficacy and life scheme subscales, the Cronbach’s alpha coefficients were 0.86 and 0.93, respectively. Table Table The main aim of this study was to translate the English version of the well-validated SIWB to Chinese and examine the psychometric properties of the Chinese version of the SIWB. The participants of this study were recruited from three different settings, namely, a hospital, a long-term care institution, and a community health center. Therefore, the results should be able to represent elderly of a diverse background. Overall, the SIWB-C showed good face validity, construct validity, and internal consistency in our study sample.Compared with the original English version of the SIBW, the SIWB-C performed well with a Cronbach’s alpha coefficient of 0.94, which is higher than the 0.91 in the English version. Two factors also emerged from the factor analysis with the same items in each factor as the English version. The Cronbach’s alpha coefficients of SIWB-C for the self-efficacy and life scheme subscales were 0.86 and 0.93, indicating high internal consistency similar to the English version. Over 68% of the total variance could be accounted for by the two-factor model and the percentage was higher than the 56% observed in the English version. In addition, the SIWB-C showed significant but moderate levels of convergent validity with EQ-5D-3 L and GDS-5, indicating these measures have unique constructs. Confirmatory factor analysis indicated a fine convergent validity of the SIWB-C and the two-factor model was well fit.The English version of the SIWB was grounded in a qualitative study of patient perspective of spirituality and well-being. It was not developed based on a religious framework. Spirituality is a universal concept, unique to all people, such as creativity, art, and self expression . The oriRegarding the translation of the SIWB-C, most items appeared to have culturally equivalent terms in Chinese and we were able to translate without the need for further adaptation. The only exception was the item 10 “In this world, I don’t know where I fit in”. We used the Chinese word for “sense of belonging” instead of “fit in” to capture the cultural connotations.The SIWB-C should be a valid and reliable instrument for use in Chinese elderly people. Nevertheless, several limitations to the current study should be noted. First, the SIWB-C was evaluated with Mandarin-speaking Taiwanese people. Its psychometric performance in other Chinese populations awaits clarification. Second, 11.5% and 11.8% of the participants were illiterate or with an educational level below primary school, respectively. Although the questions were read aloud and explained by a trained research nurse, it is possible that their responses might be affected by their literacy level. Third, the predictive validity of the SIWB-C was not tested because of the use a cross-sectional study design. Future studies should examine these issues.The translated 12-item SIWB-C was found to be a potentially useful measure of subjective spiritual well-being in elderly Taiwanese. Its application in assessing the spiritual well-being in Mandarin-speaking elderly population warrants further investigation.


N = 176), we carefully manipulated the design of the n-Back task to target general or specific resources engaged in the ongoing consolidation process. In contrast to previous studies that employed interference tasks involving conceptual stimuli and complex processing demands, we did not find any differences between n-Back and rest conditions on memory performance at delayed test, using both recall and recognition tests. Our results indicate that: (1) quiet, wakeful rest is not a necessary prerequisite for episodic memory consolidation; and (2) post-encoding cognitive engagement does not interfere with memory consolidation when task-performance has minimal semantic and hippocampally-based episodic memory processing demands. We discuss our findings with reference to resource and reactivation-led interference theories.So far, studies that investigated interference effects of post-learning processes on episodic memory consolidation in humans have used tasks involving only complex and meaningful information. Such tasks require reallocation of general or encoding-specific resources away from consolidation-relevant activities. The possibility that interference can be elicited using a task that heavily taxes our limited brain resources, but has low semantic and hippocampal related long-term memory processing demands, has never been tested. We address this question by investigating whether consolidation could persist in parallel with an active, encoding-irrelevant, minimally semantic task, regardless of its high resource demands for cognitive processing. We distinguish the impact of such a task on consolidation based on whether it engages resources that are: (1) general/executive, or (2) specific/overlapping with the encoding modality. Our experiments compared subsequent memory performance across two post-encoding consolidation periods: quiet wakeful rest and a cognitively demanding n-Back task. Across six different experiments (total  There is general consensus that wakeful rest after learning aids retention of episodic memories in humans—being able to remember our own personal past. During the post-learning rest period, recently acquired memories spontaneously reactivate by a replay of neuronal ensembles, triggered by hippocampal activity induced during the learning experience , forgetting occurs simply by competitive replacement of one encoded item (B) by another item (C) associated to the same target (A)  exhaust general resources for cognitive processing; or (b) inhibit ongoing reactivation in specific brain areas that are otherwise engaged in consolidation-relevant activities. By virtue of this fact, there have been no reports of post-encoding tasks that do not show an interference effect when compared to post-encoding rest. Interestingly, interference studies have only used tasks comprising of complex and meaningful stimuli that, as a function of their processing demands, trigger interference. Accordingly, the possibility of eliciting interference using a task that does not involve such multiple or overlapping processing has never been tested. Would engaging in a demanding task during the post-learning period interfere with memory consolidation if it: (a) requires only general resources for cognitive processing, or (b) requires resources shared with encoding but remains hippocampus-independent?n = 2), involved numbers (1–5), and differed from traditional versions, in that it provided brief trial-by-trial feedback to prompt greater engagement and reduce task-unrelated thinking. In general, our paradigm involved post-encoding periods filled with quiet wakeful rest or n-Back task in a counterbalanced order, similar to the studies reported by Dewar and colleagues  or an n-Back task (filled delay) in a counterbalanced order, and ending with a delayed recognition memory test. We compared the effect of the rest period and the n-Back task on the consolidation of items learned prior to these delays. We hypothesized a saturation of general brain resources due to high and constant allocation of attention during the n-Back task, leading to a greater effect of interference as compared to the rest condition.Mage = 22.48, SD = 3.43), were recruited from Radboud University. Ten participants were removed from the study due to technical issues, non-conformance of protocol and low performance (d-prime) on the n-Back task (2-SD below the group average). Remaining 38 subjects were included in the analyses. After receiving written and oral instructions from the experimenter, all participants gave written informed consent in accordance with the Declaration of Helsinki. At the end of the experiment, participants received course credits or monetary compensation. This study was reviewed and approved by the Ethics Committee of the Faculty of Social Sciences of Radboud University.Forty-eight native Dutch, healthy students  with its paired word . Participants were free to adopt any strategies like creating a vivid fictional story relating the two  or by simply applying the word to qualify the picture. Subsequently, the presentation of a Likert scale “1—2—3” cued the subject to press an arrow key  on the keyboard to rate the vividness of their imagery judgment. Each trial began with the presentation of a fixation cross (0.5 s), followed first by a picture (1 s) and then its paired word (3 s). Trials were self-paced with maximum allowed response duration of 5 s.n = 2) task with numbers (1–5). Each 2-Back trial started with a random grayscale number appearing in the middle of a dark screen for a maximum of 3 s during which the subjects were to press “right” if they had seen the number two trials earlier, or “left” otherwise. However, unlike traditional n-Back tasks, the grayscale number turned green (correct) or red (incorrect) for 300 ms showing a short feedback. The purpose of the feedback was to urge participants to keep their attention focused and induce optimal performance. No score or fixation-cross was displayed on the screen to avoid any visual interference. Participants were given detailed instructions and a short practice at the beginning of the experiment to acquaint them with the button presses and demands of the 2-Back task.One of the encoding sessions was followed by a delay of 12 min filled with the n-Back .t-test with d-prime scores of rest and n-Back delay periods as the dependent variables. All results were analyzed using IBM SPSS 23, at an alpha level of 0.05.In order to ensure that we included only those subjects who executed the 2-Back task at a reliable level, we excluded all participants who performed 2-SD below the group average on the d-prime score of this task. From the associative recognition task, hit rates of each list were calculated separately by dividing the number of correctly identified “old/identical” items by the total number of items to which the subject responded. False alarm rate of each list was similarly calculated by dividing the number of “new/recombined” items incorrectly identified as “old/identical”, by the total number of “new/recombined” responses. Only confident responses were included in this process to get an estimate of primarily recollection-based memory, avoiding trials performed with guesses. The standardized difference between hit and false alarm rates resulted in d-prime scores, representing subsequent memory performance  and filled delay conditions , did not differ significantly; t(37) = 0.33, p = 0.74.Average performance on the 2-Back task reached 91% accuracy, with a d-prime of 2.33 and reaction time of 0.93 s across subjects, suggesting that the participants were properly engaged in the n-Back task during the filled delay period  to 9 min and the memory test applied was changed from recognition of word-picture pairs to free-recall of words. These changes allowed the study to be more similar to the studies conducted by Dewar et al.  and CraiMage = 21, SD = 2.19) were recruited from the Radboud University student pool. Two participants were removed from the analyses due to low n-Back performance using the same exclusion criterion as before (2-SD below mean group d-prime).Twenty native Dutch, healthy students  as dependent variables.For each wordlist, a proportional retention score was calculated by dividing the number of words recalled during delayed recall by those recalled during immediate recall. In case of perfect retention, the score was capped at 1. The immediate recall scores and the proportional retention scores of the two lists were used for the analyses. First, we ran a paired samples M = 11.05, SD = 3.29; 2-Back: M = 10.55, SD = 2.59) did not differ significantly between the two encoding blocks, (t(17) = 0.919, p = 0.37), indicating that the quality of memory encoding did not differ across the two encoding sessions. As depicted in Figure M = 0.67, SD = 0.17; 2-Back: M = 0.71, SD = 0.19) also did not differ significantly across the two delay conditions; t(17) = −0.787, p = 0.44. Average performance on the 2-Back task across subjects reached 88% accuracy, with a d-prime of 2.14 and reaction time of 0.79 s.Immediate recall scores  of the n-Back task was dynamically adjusted at each trial according to subject’s cumulative performance. Integrating such dynamic difficulty adjustment could heighten the contrast between the filled and unfilled post-encoding delay periods leading to an interference effect.Experiments 3 and 4 were replications of Experiment 1 (recognition) and 2  respectively, but the standard n-Back task was replaced with a dynamic difficulty-adjusted version, referred to as the DDA n-Back. According to our hypothesis, general resources are limited to be distributed between performing a working memory task and consolidating a set of recently acquired memories. As such a highly engaging and challenging DDA n-Back task should cause a significant reduction in memory performance as compared to quiet wakeful rest.Mage = 22.29 years, SD = 3.12) and for Experiment 4, 32 participants  were recruited from Radboud University. Ten participants were excluded on account of low performance on the DDA n-Back task or technical failures, leaving 36 participants in Experiment 3 and 24 in Experiment 4 for analyses.For Experiment 3, 38 participants  available in standard 2-Back used in Experiments 1 and 2, the dynamic difficulty-adjusted version (DDA 2-Back) had a variable speed that changed with the participant’s cumulative success at each trial. The initial and slowest speed of the task was preset at 2 s per item. When the participant’s score hit 80%, the speed increased at a rate of 0.2 s with each successful trial  until it reached the maximum threshold of 0.8 s. At peak performance, the participant only had 0.8 s to report to a 2-Back trial. The increase in the participant’s skill was therefore matched by a gradual increase in task difficulty. On the other hand, if the subject’s performance fell below 60%, the speed of the task decreased at a rate of 0.1 s and kept reducing until either it returned to the initial preset of 2 s, or performance recovered back to 60%. No changes were made when the score fluctuated between 60% and 80% as this was deemed to be the “flow zone”, where task difficulty was balanced by the subject’s skill and confidence  and DDA 2-Back  delay conditions; t(35) = 0.54, p = 0.59. Similarly, in case of Experiment 4 , mean proportional retention scores  also did not differ significantly across the two encoding blocks; t(23) = −0.64, p = 0.53  did not differ between the two encoding blocks; t(23) = 0.74, p = 0.47, indicating that the quality of memory encoding was similar.As expected, the average reaction time on the DDA n-Back task was significantly faster (Experiment 3: 0.58 s and Experiment 4: 0.68 s) than its standard counterpart used in Experiment 1 (0.93 s) and Experiment 2 . Subjects encoded a large number of faces across three encoding blocks followed by one of three delay periods: rest, 2-Back or 3-Back tasks. In line with our original hypothesis, we expected a significant reduction in subsequent memory of faces learned prior to both n-Back tasks as compared to those learned prior to rest.A possible explanation for the lack of interference effects could be that the encoding material used in Experiments 1–4 (word-picture pairs and wordlists) was quite easy to remember. Perhaps, our participants  were able to retain encoded information successfully over the n-Back delay periods by making conscious or subliminal within-list or extra-list associations. The other possibility is that the load of the n-Back task itself was not demanding enough to exhaust general cognitive resources for interference to occur. Therefore, in the following experiment, we addressed these two issues by: (1) making encoding material more challenging to retain (faces); and (2) increasing the load of the n-Back task  were recruited from Radboud University. Four participants were removed due to technical difficulties or low n-Back task performance.Forty native Dutch, healthy students .Similar to the previous experiments, one of the three post-encoding delay periods was filled with 9 min of quiet wakeful rest (unfilled delay). Two separate filled delay conditions were used in this Experiment: 2-Back and 3-Back tasks. The design of the n-Back tasks remained identical to Experiments 1 and 2 and ran for 9 min as well. DDA n-Back design was not used in this experiment as it could have caused a complete failure in carrying out the 3-Back task.For each of the 270 faces  presented on the screen, participants indicated within 5 s, whether they had seen the face during the encoding session. Participants responded with keys Q and W  on the keyboard. Subsequently, they had 3 s to indicate their confidence using keys 1–4 . As before, only confident responses (“definitely sure”) were considered for later analyses.M = 1.14, SD = 0.54; 2-Back: M = 1.21, SD = 0.45; 3-Back: M = 1.20, SD = 0.45) showed no significant difference on paired samples t-tests; rest vs. 2-Back: t(35) = −0.91, p = 0.37, rest vs. 3-Back: t(35) = −0.70, p = 0.49 and 2-Back vs. 3-Back: t(35) = 0.15, p = 0.87  but no difference in their overall reaction times was observed  were recruited from Radboud University. Three participants were removed from the analyses due to technical difficulties. Remaining 24 participants performed competently during the n-Back tasks involving numbers and faces, leaving no outliers.Twenty-seven native Dutch, healthy students  downloaded from the Chicago face-database , 2-Back: M = 1.03, SD = 0.54, Face 2-Back: M = 1.11, SD = 0.38) showed no significant differences; rest vs. 2-Back: t(23) = 0.63, p = 0.53, rest vs. faces 2-Back: t(23) = −0.30, p = 0.76, and 2-Back vs. Faces 2-Back: t(23) = −0.98, p = 0.33  was also not significantly different from the 2-Back with numbers  as revealed by a paired t-test: t(23) = 0.20, p = 0.84  manipulating different aspects of the n-Back task surrounding two critical issues in relation to memory consolidation: resources and reactivation. Our findings indicate that across different versions of the post-encoding n-Back task, subsequent memory performance did not differ from an equivalent period of post-encoding rest  and administered immediately after an incidental encoding task that also involved faces. Contrary to our expectation, but in line with the results of Experiments 1–5, the subsequent memory scores reflect that the Face n-Back task did not show any reduction in memory performance relative to when post-encoding period was occupied by rest. Several theories could account for the lack of interference effect in Experiment 6. From the perspective of the encoding modality, it can be argued that repeated presentations of the same five faces during the n-Back task did not engage the face-processing areas for a sustained period of time due to the “repetition suppression effect”  a minimal overlap with previously learned memories of capitals and calculus; and (2) do not burden the long-term memory system. In short, there is no denying that quiet wakeful rest helps the mind and body in numerous ways, but to deem it necessary for consolidation is inaccurate and impractical.SV, SK and MK were involved in data collection. SV, LF, SMD contributed to the design of the experiments. SV and SMD wrote the article with input from AT, WPM and RPCK.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This case study examined how two rowers adapted their rowing patterns following crew training as a newly formed coxless pair. The two participants were expert (double-oar) single scull-boat rowers. Performing as a crew in the coxless-pair’s sweep-boat, where each rower operates a single oar, on-the-water data were collected before and after a 6-week intensive team-training program. Rowing patterns were characterized by the horizontal oar angle, oar angular velocity and linear oar-water velocity profiles during the catch  to finish  half-cycles of the propulsive water phase. After crew training, rowers demonstrated a tighter synchronization and a closer correspondence in oar angle at the moment of catch, together with a closer matching of the evolution over time of their subsequent oar movements. Most likely due to the inherent asymmetries involved in sweep-boat rowing, the stroke rower also developed a somewhat longer-duration larger-amplitude oar movement than the bow rower. Remarkably, both rowers revealed changes in the inter-cycle variability of their individual patterns of rowing. While the initially more variable stroke rower improved the consistency of his rowing pattern over practice, the initially highly consistent bow rower on the contrary relaxed his tendency to always perform in the same way. We discuss how the crew performance changed over training and to what extent it was associated with changes in individual behaviors. Along the way we demonstrate that the often-used measure of average continuous relative phase does not adequately capture the particularities of the coordination pattern observed. Overall, the results obtained at the individual level of analysis suggest that team benefits were obtained through distinct adaptations of the rowers’ individual rowing patterns. Joint action is considered as a form of social interaction whereby individual agents coordinate their movement in space and time so as to reach a common goal . While aMore generally, in joint action tasks the individual agents’ movements are shaped both by the current needs of their collective behavior and by the singular task demands that each individual agent faces. In this light, expertise in collective behavior tasks has been considered as the capability of individual agents to identify and achieve a specific contribution e.g., , thus reFigure 1 for further details). In such a setting, perfect synchronization of oar movement, with its associated symmetrical power output, in fact results in yawing (resulting in changing direction) of the boat during each propulsive drive phase, thereby reducing overall efficiency , after participants had been involved in repeated crew coordination practices. The investigation started from the very first step of their crew training. It ended after a one-month-and-an-half intensive team practice focused on enhancing their coordinative capability. The goal of the study was to simultaneously characterize the changes in the collective and individual rower behaviors.Pôle Espoir Aviron – Nantes), both were qualified as expert-level individual rowers. Each rower had more than 10 years of experience in single scull (two-oar) rowing. Rowers individually performed in the national competition and belonged to the French top 10. While both had rowed in crew boats during training sessions, neither had experienced dedicated crew training. Before engaging in the present study they had never rowed together in the same boat.Two 17-year-old men participated in the study. Having been admitted into the French National Rowing School  that took place before and after a 6-week training program dedicated to crew rowing. We will refer to these two data-collection sessions as pre-CT and post-CT, respectively. The intensive CT program was managed by the national coach and comprised 26  on-water practice sessions, for a total of almost 50 h of coxless pair rowing practice. Each practice session typically consisted of two sets of 20–30 min of rowing separated by 5-min rest periods. During training sets, performed at frequencies of 17–28 strokes per minute (spm), rowers had to use maximal power during the drive , so as to move the boat forward as fast as possible, and to recover when the oar was out of the water. During practice sessions, the coach followed the coxless pair in a motorboat, providing online feedback mainly focusing on the simultaneity of the oars’ entry into the water, the orientation of the blades and the direction of the boat. Crew briefings providing further information were organized before and after each CT training session.The pre-CT and post-CT data-collection sessions took place under calm water and stable weather conditions while rowing at constant pace of 17–18 spm under the same general instructions as described for the training sessions. Both rowers had extensive previous individual practice experience at this stroke rate. Moreover, it did not induce a level of fatigue that could be expected to alter the rowing patterns over the course of the approximately 20-min sessions during which data was collected.The study was performed in accordance with the Declaration of Helsinki and the APA ethical guidelines. It was approved by an Institutional Review Board of the University of Nantes. The two rowers and their coaches were informed of the procedures. The rowers, their parents and the staff members in charge provided written informed consent. Both sessions analyzed in the present study were part of a larger research project (ANOPACy), also including qualitative phenomenological analyses of the experience of crew rowing, using individual rower verbalizations obtained during video-based self-confrontation interviews  and otheDuring the pre-CT and post-CT sessions, behavioral data were collected at 50 Hz using the PowerLine system . For the present purposes, we retained the time series of horizontal oar angles (delivered by position sensors in the oarlocks) and boat velocity (delivered by an impeller fixed under the shell). According to Figure 2.Full time series of the 350 recorded strokes were first filtered using a low-pass Butterworth filter with a 7-Hz cut-off frequency, run through twice in order to negate the phase shift. Oar angular velocity (OAV) time series were subsequently derived using the first central difference method. The first 10 cycles were then removed in order to focus on stabilized performance, leaving 340 full strokes for analysis purposes. Samples of five subsequent strokes from the pre-CT and post-CT sessions are presented in entry (where the blade enters the water), drive (where the blade drives the boat forward), release (where the blade exits the water) ending at the finish point, and recovery from the finish to the next catch point  for each rower separately. Starting from the catch point, a rower’s full stroke is defined by four subsequent phases: ch point , p. 45.In order to quantify individual rower behavior, we extracted for each rower at each session the time series of oar angle and OAV of the half-cycles between catch and finish points. Each of these 340 half-cycles was time-normalized using steps of 2% half-cycle duration, resulting in 51 points per half-cycle. Average time-normalized half-cycles for each rower at each session were then obtained for oar angle and OAV by calculating the mean of all 340 corresponding values at each of the 51 points. The variability over half-cycles was calculated as the standard deviation over all 340 corresponding values at each of the 51 points.As we were mainly interested in the (propulsive) drive phase, we identified this phase by determining when the oar moved faster than the water. To this end, for each of the 340 extracted half-cycles of each rower in each session, we determined the linear oar velocity in the direction of the boat’s longitudinal axis by multiplying the tangential oar velocity (defined by the product of OAV and oar length) with the cosine of the oar angle. By calculating the difference between instantaneous linear oar velocity and instantaneous boat velocity and averaging over the 340 cycles, we obtained average time-normalized half-cycles of linear oar velocity with respect to the water [linear oar-water velocity (OWV)].Figure 3, the drive phase (shaded areas under the curves where linear OWV is positive) ended closer to the finish point for the bow rower than for the stroke rower, at least during the pre-CT session. In order to compare the behavior of individual rowers on and between sessions, for both rowers we therefore selected the common (29-point) period from point 14 to point 42  for analyses of the drive phase.As can be seen from Figure 3) it is important to realize that these individual catch and finish points did not necessarily coincide in time, as becomes clear from inspection of Figure 2. In order to capture the collective behavior of the two rowers, we therefore extracted the 340 synchronous oar angle and OAV time series of the rowers during each (catch-to-finish) half-cycle of the stroke rower. After time-normalization to 51 points according to the procedure described above, for each of these 340 time-locked series we quantified between-rower coordination by the continuous relative phase (CRP) between the motions of the two oars during the catch-finish half-cycles. To this end, for each rower and each half-cycle, oar angle was normalized to a  interval based on the minimal and maximal oar angles  and OAV was normalized by dividing OAV by peak OAV (resulting in a maximum of +1); Phase was determined as the angle (clockwise notation) formed by each point thus defined in the normalized phase plane. CRP was defined at each point as the difference between the phases of the stroke and bow rowers . In examining the resultants plots  were performed using t-tests over the 29 points defining the drive phase. Paired tests were used for within-rower comparisons (pre-CT vs. post-CT for bow and for stroke) while independent sample tests were used for between-rower comparisons (bow vs. stroke at pre-CT and at post-CT). Because only one pair of rowers was considered in the present study, significant (α = 0.05) effects were only considered when effect size (Cohen’s d) reached at least the 0.50 threshold for a medium size effect  lower stroke rate during the post-CT session was accompanied by a 2.2% lower average boat velocity .Figure 2, durations of catch-to-finish half-cycles were shorter than durations of the complementary finish-to-catch (recovery) half-cycles. During the pre-CT session the durations of the catch-to-finish half-cycles were 1.016 ± 0.046 s and 1.152 ± 0.111 s for the bow and stroke rower, respectively; during the post-CT session the corresponding durations were 1.057 ± 0.053 s and 1.118 ± 0.073 s, respectively. The difference in (catch-to-finish) half-cycle durations thus decreased over practice (from 0.137 to 0.061 s), but remained significant at the time of the post-CT session, d = 0.69.As can be already be seen in d = 3.46. During the pre-CT session peak angular velocity was slightly lower for the stroke rower (115.1 ± 3.3 deg/s) than for the bow rower (117.9 ± 3.3 deg/s), d = 0.81. This difference no longer existed during the post-CT session .While amplitudes of oar displacement from catch to finish were almost identical during the pre-CT session , a difference came to the fore during the post-CT session, mainly due to an increase in amplitude for the stroke rower , during the pre-CT session the bow rower demonstrated a particularly consistent pattern of oar angular (OA) displacement during the drive phase, with an OA variability (defined as the 29-point average of standard deviations over the 340 drives) of 1.80 ± 0.31°. The stroke rower’s movements during this pre-CT session were considerably more variable (d = 3.81), with an OA variability of 6.15 ± 1.59°. Interestingly, over practice not only the stroke rower’s OA variability decreased , but the bow rower’s OA variability increased . While the difference between individual rower OA variabilities thus decreased over practice, it remained significantly lower for the bow rower, d = 1.49.As can be seen from Figure 5) during the drive phase. During the pre-CT session, the OAV variability was smaller for the bow rower (4.75 ± 0.40°) than for the stroke rower (9.03 ± 7.81°), d = 0.77. Both rowers decreased their OAV variability over practice, reaching 4.01 ± 0.59° for the bow rower (d = 1.35) and 5.68 ± 3.75° for the stroke rower (d = 0.73) during the post-CT session. Although the difference between individual OAV variabilities decreased over practice, it remained significantly lower for the bow rower, d = 0.62.A slightly different pattern of results emerged for the variability in OAV  revealed a smaller OWV variability for the bow rower (0.21 ± 0.04 m/s) than for the stroke rower (0.47 ± 0.39 m/s) during the pre-CT session, d = 0.93. Both rowers decreased their OWV variability over practice, reaching 0.18 ± 0.05 m/s for the bow rower (d = 0.95) and 0.28 ± 0.21 m/s for the stroke rower (d = 0.99) during the post-CT session. Although the difference between individual OAV variabilities decreased over practice, it remained significantly lower for the bow rower, d = 0.63.Applying the same analysis to the linear OWV  decreased from the pre-CT session (4.95 ± 2.38°) to the post-CT session (2.23 ± 1.77°), d = 1.30, indicating an increase in space-time similarity of the oar movements of the two rowers. Perhaps surprisingly, the nature of the between-rower coordination appeared to change as average CRP  evolved from -0.30 ± 4.44° (pre-CT) to -4.09 ± 3.86° (post-CT), d = 0.91, suggesting the coming to the fore of a phase lag of the stroke with respect to the bow rower. Inspection of Figure 2, however, suggests that, contrary to what is generally assumed (d = 1.57). Such a change was not observed for the timing of the finish, with the stroke-bow difference being 0.065 ± 0.105 pre-CT and 0.064 ± 0.081 post-CT, d = 0.01. Thus, during the pre-CT session, the bow rower entered the water somewhat before the stroke rower and left the water somewhat after the stroke rower. Over practice this timing difference disappeared for the catch, with both rowers entering the water at the same time after training, but not for the finish (release). The timing of the catch was not the only aspect that changed over practice; the position of the oars  at catch and finish also evolved. The stroke-bow difference in oar angle decreased for the catch, from 4.18 ± 1.67° pre-CT to 1.79 ± 1.39° post-CT (d = 1.56), while the stroke-bow difference in oar angle increased for the finish, from 4.67 ± 1.36° pre-CT to 7.34 ± 0.90° post CT (d = 2.32). As can be seen from Figure 2, these results indicated that over practice the two rowers came to adopt similar oar angles when entering the water, while accentuating their difference  when leaving the water.At the collective level, the RMS difference between oar positions  becoming more tightly matched between the two rowers in terms of both timing and oar-angle magnitude. However, the results also suggested a subtler change in the nature of the coordination of the rowers’ oar movements, with the stroke rower, developing an oar movement of a longer duration, continuing up to a larger amplitude than the bow rower in the post-CT session. This finding might be interpreted as the crew’s solution to  avoid channeling the boat into yawing during the drive phase: full space-time similarity of the rowers’ oar movements in the asymmetrically rigged sweep-boat would indeed result in differences in the moments produced by each rower . We notevariability of the kinematic patterns of oar movement . On the post-CT session the stroke rower demonstrated increased consistency  over the drive phase on all three measures. While the bow rower also improved his consistency for OAV and linear OWV, he revealed an increase in variability in oar angle displacement during the drive phase.The foregoing discussion already brings out that results observed at the level of the individual rowers consolidated and enlightened the idea that improvement in crew behavior was rooted in changes in how each rower performed his own movement. Interestingly, apart from the results on oar movement amplitude and timing alluded to above, we also observed training effects at the level of the The finding that, at the level of oar angular displacement, crew training resulted in a decrease of inter-cycle variability for one rower and an increase for the other rower is particularly noteworthy, as it speaks to the adaptability of individual patterns. Following training, the  stroke rower performed in a more stable manner, while the bow rower relaxed his initial tendency to always perform in the same way. Interestingly, this result highlights how a team member can change his behavior in terms of reducing its absolute efficiency  in order to obtain benefits at the team scale. Although we do not have direct proof for this, we suggest that the bow rower became better coupled to the stroke rower, enhancing the process of reciprocal compensation  that supwhen of each rower’s oar movement), but also required each rower to change the how of the rowing movement. The training effects observed here complement findings from other domains . We described how crew rowing changed over training and the extent to which it was associated with related changes in individual behaviors. Among the key results, our case study suggested the capability to change individual patterns of rowing as being the key element underlying the positive collective behavioral transformation. Our results also proposed individual variability of behavior as being an important variable to consider, while the need to either decrease or increase it to obtain team benefits may be task-dependent. In terms of the questions that remain open, the extent to which individual signatures converge or diverge through team training similarities) should be further investigated in future research.MF, MR, and JB conceived, designed and ran the study. MF, MR, RB, and JB analyzed and interpreted the data. All authors participated drafting the work and/or revising it critically for important intellectual content. The final version submitted was approved by all authors.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer RG and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.


Routine clinical practice data often differ from clinical trials. This study describes real-world treatment patterns and effectiveness among patients with metastatic colorectal cancer (mCRC) receiving ziv-aflibercept in non-academic, community oncology practices in the USA. De-identified electronic medical records from Vector Oncology and Altos Solutions databases were analysed. We identified 218 patients diagnosed with mCRC who had received prior oxaliplatin therapy and initiated ziv-aflibercept as part of second-line or later-line therapy. Overall survival (OS) and progression-free survival (PFS) were estimated using Kaplan–Meier analysis. Mean age was 62.8 years at ziv-aflibercept initiation. Most patients (91.7%) received bevacizumab before ziv-aflibercept, 95.4% initiated ziv-aflibercept with FOLFIRI or another irinotecan-based regimen, and 59.6% had received prior irinotecan. Overall, 24.8% of patients initiated ziv-aflibercept in second line, 31.7% in third line, 21.6% in fourth line and 22.0% in later lines of therapy. Mean duration of ziv-aflibercept treatment was 5.3 months. For patients initiating ziv-aflibercept in second-, third- and fourth-line therapy, median OS was 11.9 , 11.1 (6.9–16.7) and 8.1 (5.2–11.4) months, respectively, and median PFS was 4.4 (2.8–6.5), 4.3 (2.9–6.3) and 3.4 (2.2–5.2) months, respectively. Common adverse events (AEs) (any grade) included gastrointestinal disorders (64.7%) and asthenia/fatigue (63.3%). In routine clinical practice, ziv-aflibercept was frequently initiated in third line or later lines of therapy. Although patients receiving ziv-aflibercept were more heavily pretreated and potentially less robust compared with the VELOUR trial, median OS for patients receiving second-line ziv-aflibercept was comparable. AE rates were similar to or lower than the VELOUR trial. Colorectal cancer (CRC) is the fourth most common cancer in the USA, with 132,700 new cases and 49,700 deaths estimated to occur in 2015 . The study protocol was reviewed and approved by the New England Independent Review Board. Because of the retrospective nature of this study, patient informed consent was not required in accordance with institutional regulations.Eligible patients had mCRC and had received ziv-aflibercept (second line or later) not as part of a clinical trial. Patients were required to have received oxaliplatin therapy prior to ziv-aflibercept initiation. In addition, patients were required to have no other primary tumours during the baseline period (1-year period before ziv-aflibercept initiation).The date of ziv-aflibercept initiation was defined as the index date. The follow-up period started at the date of ziv-aflibercept initiation and ended at the data cut-off date, last clinic visit date or death. Baseline clinical characteristics were assessed before or at the index date of ziv-aflibercept initiation, and the distribution of the line of ziv-aflibercept initiation was described. Lines of therapy after mCRC diagnosis were identified using the following algorithm based on chemotherapy/targeted therapy administrations. The start of first-line therapy was defined as the first administration of chemotherapy or targeted therapy after diagnosis of mCRC. The first-line regimen contained all chemotherapy-related drugs administered within 28 days of the start of the regimen. Subsequent lines of therapy were identified by the first administration of any chemotherapy-related drug not used in the previous line of therapy. The subsequent regimens contained all chemotherapy-related drugs that were administered within 28 days of the first administration in that line of therapy. If there was a gap of ≥ 120 days between treatment administrations within a given line of therapy, the second treatment was considered a new line of therapy. Chemotherapy or targeted therapy administrations were identified based on review of generic names of administered medications, review of generic names for oral medication prescriptions and information for oral chemotherapy medications. The end date for each line of therapy was defined as the last date of administration of any of the drugs used in the regimen before the start of the subsequent line of therapy. Duration of ziv-aflibercept treatment was measured in days from the first administration of ziv-aflibercept to the last administration of ziv-aflibercept. Duration of the chemotherapy regimen containing ziv-aflibercept was measured in days from the first administration of the first drug administered in the regimen to the last administration of any of the chemotherapy drugs used in the regimen before the start of the subsequent line of therapy. Patients were considered censored if the last administration of either the ziv-aflibercept or the concomitant chemotherapy regimen was within 60 days of the date of last contact. Reasons behind discontinuation of ziv-aflibercept and the concomitant regimen, reasons for treatment interruptions and dose changes, and the date of last contact/death were also collected.Other outcomes data  were also assessed. Objective tumour response was based on information available in the EMR notes; to the extent information was available, tumour response was further classified as complete or partial. OS was defined as the time from ziv-aflibercept initiation to death or last contact (for censored patients). PFS was defined as the time from ziv-aflibercept initiation to first disease progression or death (whichever occurred earliest) or last contact (for censored patients). Information about treatment-emergent AEs during ziv-aflibercept treatment was extracted for prespecified events, including those reported in the ziv-aflibercept prescribing information. For resource utilization, the number of patients with hospitalizations and emergency room visits after ziv-aflibercept initiation was reported.Statistical analysis was performed on the Altos Solutions and Vector Oncology datasets, both individually and on the pooled data. Data extracted from the individual databases are reported for baseline patient and clinical characteristics in this publication; remaining data and analyses are shown for the pooled dataset.To assess the heterogeneity of the populations from the two data sources, meta-analysis random effects models were estimated for select baseline characteristics, including age, gender, race, tumour location, number of sites of metastases, occurrence of liver metastases, presence of hypertension, months from first mCRC diagnosis to ziv-aflibercept initiation, and prior bevacizumab use .Continuous variables were summarized by mean, median, standard deviation and interquartile ranges (or minimum and maximum). Categorical variables were summarized by absolute frequencies and percentages. Kaplan–Meier analyses were performed to evaluate time to discontinuation of the entire chemotherapy regimen concomitant with ziv-aflibercept, time to ziv-aflibercept discontinuation, PFS and OS. Survival rates at 6 and 12 months were also reported. These variables were analysed both in the overall population and according to the line of therapy at which ziv-aflibercept was initiated. All data manipulations and statistical analyses were performed using SAS version 9.3 software.n = 107; Altos Solutions database, n = 111). The mean age at ziv-aflibercept initiation was 62.8 years (Table p < 0.05) heterogeneity between populations for most baseline characteristics except months from mCRC diagnosis to ziv-aflibercept initiation and prior bevacizumab use.In total, 218 patients were included in the final study population , 46.4% had wildtype KRAS and 53.6% had a KRAS gene mutation; among patients with biomarker data for BRAF (n = 46), 82.6% had wildtype BRAF and 17.4% had a BRAF gene mutation. The most common site of metastasis was the liver (79.4%). The most frequent conditions diagnosed prior to ziv-aflibercept initiation were hypertension (55.5%) and tobacco use (29.8%).In patients with available biomarker data for KRAS , third-line (n = 69) and fourth-line (n = 47) treatment, 25.9, 20.3 and 21.3% achieved complete or partial response, respectively, and 14.8, 21.7 and 19.1% achieved stable disease.Effectiveness among patients treated with ziv-aflibercept in routine clinical practice was evaluated in terms of best tumour response, PFS and OS Fig. . OverallThe estimated 6-month OS rate following ziv-aflibercept initiation was 65.1% (95% CI 58.1–71.2) and the estimated 12-month OS rate was 42.7% (95% CI 35.4–49.7). At the end of study follow-up, 35.3% of patients remained alive and the median OS was 9.6 months (95% CI 7.5–11.9) , asthenia/fatigue 63.3%), thrombocytopenia (27.1%), neutropenia (25.7%), hypertension (24.3%), infection (23.4%) and decreased appetite (22.0%) . In the VELOUR study, ziv-aflibercept was administered as part of second-line treatment in all patients. In contrast, in this real-world analysis only 24.8% of patients initiated ziv-aflibercept as second-line therapy, more than two-thirds of patients initiated ziv-aflibercept as third-line or later-line therapy, and six patients (2.9%) initiated ziv-aflibercept in the seventh line or later. Also, nearly 60% of patients in this analysis received prior irinotecan-based therapy compared with the VELOUR trial, which required patients to be irinotecan-naïve. In addition, over 90% of patients in this analysis received bevacizumab prior to the initiation of ziv-aflibercept, compared with only 30% of patients enrolled in the VELOUR trial .Despite patients in this analysis being more heavily pretreated—and potentially less physically fit—compared with the VELOUR trial, the median OS for patients receiving second-line ziv-aflibercept was approximately 12 months for both studies . The medThe rate of treatment-emergent AEs experienced by patients in this analysis was similar to or lower than the rates observed in the VELOUR trial. In the VELOUR trial, the most commonly experienced AEs were anaemia , diarrhoea , neutropenia , proteinuria  and asthenic conditions  [We acknowledge that these findings should be interpreted with caution; data generated from an analysis of patients treated in routine clinical practice and from clinical trials cannot be directly compared because of differences in patient populations. As a study using EMR data, this analysis has several limitations. Data fields such as clinical characteristics are not required for reimbursement and hence are often not well populated. Also, prescription data  are not as reliable as administration data for injectable/infused chemotherapy agents. To mitigate these concerns and obtain more complete and accurate data, this study supplemented the structured EMR data with a review of unstructured EMR records from the Altos Solutions and Vector Oncology databases. Also, the determination of chemotherapy regimens and lines of therapy from individual administrations of chemotherapy and targeted agents is sensitive to the specific algorithm that is used. Therefore, an algorithm with different rules may yield different results. In addition, the overall sample size and follow-up for this study was limited by the relatively recent approval of ziv-aflibercept in 2012 . TreatmeIn summary, this study evaluated routine clinical practice data of patients with mCRC who initiated ziv-aflibercept as second-line or later-line therapy following a prior oxaliplatin-based treatment regimen. Ziv-aflibercept was frequently initiated in the third-line or later-line setting during the first 2 years after drug approval in the USA. Although patients treated with ziv-aflibercept in our cohort were typically more heavily pretreated, and potentially less robust, than in the VELOUR clinical trial, the median OS was similar to VELOUR and also to other previously published trials of second-line anti-VEGF therapy in mCRC , 7, 9. T


To assess the accuracy of contrast-enhanced ultrasound (CEUS)-CT/MR image fusion in evaluating the radiofrequency ablative margin (AM) of hepatocellular carcinoma (HCC) based on a custom-made phantom model and in HCC patients.n = 6) and an incomplete ablation group (n = 18). After radiofrequency ablation (RFA), the AM was evaluated using ultrasound (US)-CT image fusion, and the results were compared with the AM results that were directly measured in a gross specimen. CEUS-CT/MR image fusion and CT-CT / MR-MR image fusion were used to evaluate the AM in 37 tumors from 33 HCC patients who underwent RFA.Twenty-four phantoms were randomly divided into a complete ablation group  and 13.5 ± 4.5 min (range: 8–16 min) for CEUS-CT/MR and CT-CT/MR-MR, respectively. The sensitivity, specificity, and accuracy of CEUS-CT/MR imaging for evaluating the AM were 100.0, 80.0, and 90.0%, respectively.A phantom model composed of carrageenan gel and additives was suitable for the evaluation of HCC AM. CEUS-CT/MR image fusion can be used to evaluate HCC AM with high accuracy. Radiofrequency ablation (RFA) is a radical treatment for hepatocellular carcinoma (HCC) and has a relatively low risk , 2. HoweIn recent years, novel medical imaging methods have been explored to assess AM in HCC patients after ablation, including CT-CT image fusion , 22, MR-In this study, we established a phantom model to evaluate AM based on US-CT image fusion. The aim of this study was to assess the accuracy of CEUS-CT/MR image fusion for the evaluation of the AM of liver tumors, both in an in vitro phantom model and in the clinic. The gross specimen of the phantom after RFA was used as a gold standard. The clinical AM results obtained using CEUS-CT/MR image fusion and MR-MR/CT-CT image fusion were compared.The materials used to construct the phantom model included carrageenan  and a number of additives such as oral ultrasonic contrast agent , gastric window contrast agent , milk, and Congo red.The phantom model included a spherical tumor model  in which the ablative area was covered by AM and an incomplete ablation group (n = 18) in which the ablative area was not covered by AM.A total of 24 phantom models were randomly divided into two groups, including a complete ablation group  using a 17-gauge, internally cooled-tip electrode with a 3-cm tip. A MyLab Twice ultrasound machine  and a linear probe LA332 (frequency range from 3 to 11 MHz) with imaging fusion  and three-dimensional software were employed for ultrasound guidance and exploration.An ablative area model was established by ablating the tumor model. Electrodes were inserted into the cylindrical model parenchyma via ultrasound guidance by an experienced ultrasound interventional doctor. The RFA was set in impedance mode with maximum output. According to our pilot studies, the duration of ablation was 4 and 6 min for the incomplete and complete ablation groups, respectively. After ablation, the liver tumor model together with the AM model and a portion of the parenchyma model were melted and mixed. An ablative area model was established after cooling and solidifying the melted gel.Each phantom CT scan was performed without contrast medium prior to ablation. The CT scan was performed using a 64-row multi-detector CT scanner . The following CT parameters were applied to acquire dynamic data: 1-s gantry rotation time, 120 kV, 80 mA, acquisition in 264 transverse mode (64 sections per gantry rotation), and 2.5-mm reconstructed section thickness  ultrasound unit and convex array transducer CA431 (4–10 MHz) 10–15 min after ablation. Virtual Navigator was the image fusion program and CnTI (MI <0.05) was the imaging technique for contrast-enhanced ultrasound in the ultrasound unit. SonoVue  was used as the contrast agent. For each application, 2.4 ml of SonoVue was administered through the antecubital vein and flushed by 5 ml of normal saline.The method of CEUS- CT/MR image fusion used in this study had been reported in our former article . The CT/CT-CT/MR-MR image fusion if the CT/MR at 1 month after RFA revealed that the lesion was completely ablated.Contrast-enhanced CT/MR was performed 1 month before and after the RFA for all patients. AM was further evaluated using One CT/MR portal or delayed phase series with a clearly demonstrated hepatic vessel and ablative area before RFA in DICOM format was transferred into the navigation system in MyLab Twice. One month after RFA, another series of CT/MR images were also imported into the image fusion system. The system then automatically displayed six pictures in two rows: the upper row included the transaction, coronal and vertical section CT/MR images before RFA, and the lower row showed the corresponding CT/MR images after RFA. The HCC lesion in the CT/MR before RFA was manually outlined, and then a 5-mm AM was set automatically in different colors Figs. , 4b.Image registration was performed by aligning two overlaid CT/MR images. Translation and rotation were performed in three reformed planes to maximize the image similarity around the HCC lesion and the area of ablation. The hepatic vein, hepatic artery portal complex and hepatic contour near the lesion were used as landmarks for fine adjustments to obtain a satisfactory registration. The pre- and post-RFA CT/MR images were then overlapped to assess whether the ablative area encompassed the HCC lesion and the 5-mm AM. The standards of complete registration included complete matching of three corresponding anatomic landmarks adjacent to the tumor, and the offset was less than 5 mm in each plane. Failed registration was determined when the above standards were not achieved after three attempts. The time spent on registration for each lesion and the success rate of MR-MR image fusion were recorded. The results of the CT-CT/MR-MR image fusion as standard were used to evaluate the accuracy of the CEUS-CT/MR image fusion.The analyzed data included 1) the duration required for the US-CT image fusion (phantom model study), CEUS-CT/MR image fusion, and MR-MR image fusion ; 2) the success rate of US-CT image fusion (phantom model study), CEUS-CT/MR image fusion and CT-CT/MR-MR image fusion ; 3) the accuracy rate of the assessment, including the coincidence rates of the assessment of complete ablation between US-CT image fusion and the gross specimen (phantom model study), and between CEUS-CT/MR image fusion and CT-CT/MR-MR image fusion; and 4) the maximum thickness of the residual AM in the US-CT image fusion and the gross specimen.t test was used to compare the maximum thickness of the residual AM in the US-CT image fusion and the gross specimen. A P value less than 0.05 was considered significant.Statistical analyses were performed using SPSS for Microsoft Windows . The data are the mean ± standard deviation (range). The paired The echogenicity, density, and color of different components of the phantom models met the requirements Table . The appP = 0.705).Of the 24 phantom models, one phantom was accidentally damaged, and 23 phantoms were used in all follow-up experiments. Image fusion was successfully obtained from the 23 phantoms. The success rate of image fusion was 100% (23/23). The average time used for image fusion was 5–12 min (median = 7 min). Compared with the gross specimen, the sensitivity, specificity, and accuracy of the US-CT image fusion for the detection of residual AM were 100.0, 93.8 and 95.7%, respectively . The duration was 8.5 ± 2.8 min range: 4–12 min) and 13.5 ± 4.5 min (range: 8–16 min) for the CEUS-CT/MR and CT-CT/MR-MR image fusions, respectively. The results of the AM evaluation based on CEUS-CT/MR and MR/MR image fusions are shown in Table –12 min aPhantoms have been widely used to evaluate the effects of thermal treatments. Previous studies, however, have mainly focused on other topics, such as temperature monitoring, energy distribution, the relationship between RF and electrical conductivity, and development of the heating algorithm applied in drug delivery. It is not clear whether phantoms are good models for the evaluation of AM using a CEUS-CT/MR image fusion system. Therefore, in the present study, we established a phantom model to evaluate AM using CEUS-CT/MR image fusion. In our study, we found that the peculiarly thermal invertibility and thermal sensitivity of the carrageenan gel were useful for distinguishing the ablative zone, the remaining ‘tumor’, and the AM after RFA. While the carrageenan hybrid gel used in the present study was not the best material for the evaluation of thermal ablation, especially for temperature variation and energy distribution, we took full advantage of the physical properties of the carrageenan gel. To the best of our knowledge, this is the first report to assess the accuracy of the evaluation of complete RF using an image fusion system that matched pre-RFA and post-RFA images in a tissue-mimicking phantom.We developed a hybrid gel phantom using carrageenan and other substances, which have a number of important properties, i.e., sufficient strength, low fragility, and low cost. Carrageenan, a high-molecular-weight polysaccharide extracted from red algae, consists of repeating galactose and 3,6-anhydrogalactose units linked by alternating α-1,3- and β-1,4-glycosidic linkages. Carrageenan can be used in a phantom model because it is inexpensive and safe, as well as broadly applied for the production of gel products and other foods. Additive agents played significant roles in the construction, imaging, and observation by the naked eye. For example, NaCl was added to the carrageenan gels to adjust the gel conductivity. US contrast agent and iodipin were used to improve the echo or to enhance attenuation. In addition, Congo red, an indicator used for the diagnosis of amyloidosis by generating a bright and distinct red color, was easily distinguished from the opaque gel. The red color may have infiltrated the peripheral gel due to the diffusion of Congo red. However, we believe that Congo red has no influence on the results of the AM evaluation if the whole procedure, including manufacturing, RFA, and assessment of the ablative zone, is completed within 6 h. Using carrageenan together with other substances, we were able to create a large and robust phantom model with excellent shape retention. The turbidity and low fragility of the carrageenan gel in the phantom model ensured accurate image registration. In addition, we designed a phantom that mimicked the tumor lesion  to assess the AM using the fusion imaging system after RF. The easy heating and coagulation of the phantom model allowed us to assess the post-RFA destructive zone more accurately. Improved visualization of the target by US and CT, as well as the distinct color of the materials, also improved the ablation assessment. Therefore, the phantom model established herein was successfully used for the evaluation of the AM of the HCC tumor.The present experimental study results suggest that the US-CT fusion image system can be used to accurately and effectively evaluate AM. However, in one case, US-CT image fusion revealed that the AM was completely covered by the ablative area, and even less than a 1-mm AM was observed in the gross specimen. The false-positive case could be caused by registration error and magnetic positioning system error. Given that the phantom model was idealized to evaluate AM, the feasibility and accuracy was further validated in a clinical assessment.In our clinical study, the sensitivity, specificity, and accuracy of CEUS-CT/MR image fusion for the evaluation of AM were 100.0, 80.0 and 90.0%, respectively, suggesting that CEUS-CT/MR image fusion is a good tool for evaluating AM after HCC ablation. CEUS-CT/MR image fusion combines the advantages of CEUS and CT/MR and expands the use of both imaging methods, including the high spatial contrast resolution of CT/MR and real-time guidance, accessibility, and practicality of ultrasound. In addition, CEUS-CT/MR image fusion greatly improves intraoperative AM evaluation and the localization of tumor lesions compared with MR-MR image fusion.We discovered three false-positive cases in the clinical study, which might be due to the ability of CEUS to only demonstrate blood perfusion of tissues rather than necrosis. The high temperature of the local zone of ablation may cause swollen tissues and small vessel occlusion, limiting the infiltration of blood into the ablated area. However, occluded small vessels can be reperfused after the local tissue temperature decreases, suggesting that the ablation zone may be over-measured by intraoperative CEUS.The present study has several limitations. First, the phantom model cannot completely mimic dynamic tissues and organs, such as respiratory movements, which may reduce the accuracy of the registration for image fusion and affect the imaging assessment. While the assessment was performed successfully in the idealized phantom model, some unknown problems may be present in the in vivo experiments, which must be identified and resolved. Second, the phantom models applied in the present study were used for US-CT image fusion, whereas most of the clinical cases were evaluated by CEUS-CT/MR image fusion. Thus, the accuracy of the results may be biased. Third, all of the patients enrolled in this study were male, which may also bias the results. Therefore, further studies with more experience, a larger sampling size and better technology are needed.In conclusion, we successfully established a phantom model for the evaluation of AM using US-CT/MR image fusion. Our results suggest that US-CT/MR image fusion is an accurate approach for evaluating AM after tumor ablation based on both an in vitro model and a clinical study.